Ionomics-metabolome association analysis as a new approach to the impact of dietary copper levels in suckling piglets model

Ionomics-metabolomics association analysis is a novel method to elucidating the potential mechanisms underlying the effects of dietary copper on the overall health parameters of suckling piglets model. Few studies have elucidated the relationship between the changes of ionic and metabolic homeostasis responses to dietary copper level. The growth performance data was obtained from 180 suckling piglets which access to different copper levels: 6 (low copper diet, LC), 20 (control diet, CON), and 300 (high copper diet, HC) mg·kg−1 copper (based on diet, supplementation from CuSO4), and offered ad libitum from d 14 until weaning at 40 d of age. Dietary high level copper (300 mg·kg−1) increased the ADG and ADFI during d 14 to 28 of piglets. Six elements (Mg, Na, K, P, Cu, and Mn) concentrations significantly changes in hair among the three treatment diets. The significant increased concentrations of Na and K, and decreased concentration of Mg and Mn in 300 mg·kg−1 than 20 mg·kg−1 copper diet was observed. In current study, with the increase in copper level from 20 to 300 mg·kg−1 in diet, the correlation between hair Na, K and Cu, Mn, Zn vanish. Hair Na and K were positively correlated with serum total antioxidant capacity (T-AOC) and negatively correlated with tumor necrosis factor-α (TNF-α). The hair Cu was negatively correlated with serum malondialdehyde (MDA), total bile acid (TBA). The fecal Cu was positively correlated with serum growth hormone (GH). The results suggested that the average daily gain (ADG) in 6 mg·kg−1 copper diet and the average daily feed intake (ADFI) in 20 mg·kg−1 copper diet were decreased than 300 mg·kg−1 copper diet during d 14 to 28 and the ADG was decreased in 6 and 20 mg·kg−1 copper diets in d 29 to 40 of piglets. Dietary 20 mg·kg−1 copper maintain ion homeostasis due to increase the number of positive correlations between macroelements-microelements in hair and serum. Significantly changed Na, K, Mg, Mn and Cu concentrations in hair can reflect the adverse effects of dietary 300 mg·kg−1 copper of suckling piglets. We believe our results may benefit people to gain a better understanding of the ion interactions and metabolic homeostasis of heavy metal elements that are critical to human and animal health.

Ionomics profiles. Thirteen elements concentrations in hair, serum, and feces were described in Fig. 1. In hair, compared with piglets fed the CON diet, Mg, P, and Mn concentrations were decreased (P < 0.05) in HC diet, the Na, and K concentrations were increased (P < 0.01) in HC diet. Compared with piglets fed the LC diet, the Cu concentration was increased (P < 0.01) in HC diet (Fig. 1a-c). In serum, compared with piglets fed the LC diet, the Mg concentration was increased (P < 0.05) in CON diet, and the P concentration was increased (P < 0.05) in HC diet (Fig. 1d). No significant differences of Fe, Cu, Mn, Cr, Pb, Al, and Ni concentrations among each diet were observed (P > 0.05) (Fig. 1e,f). In feces, compared with piglets fed the LC diet, the Cu concentration was increased (P < 0.01) in HC diet (Fig. 1h). No significant differences of Ca, Mg, Na, K, P, Pb, Ai, and Ni concentrations among each diet were observed (P > 0.05) (Fig. 1g,i).

Correlation between ionomics profiles and serum biochemical parameters. The correlation
between ionomics profiles and serum biochemical parameters were described in Fig. 2. Serum GH was positively (P < 0.05) correlated with fecal Cu and Zn; serum TNF-α was negatively (P < 0.05) correlated with hair Na and K and positively (P < 0.05) correlated with fecal Cr; serum MDA was negatively (P < 0.05) correlated with hair Fe, Cu, and Mn and positively (P < 0.05) correlated with fecal Fe, Cr and Pb; serum T-AOC was positively (P < 0.05) correlated with hair Na and K and negatively (P < 0.05) correlated with fecal Mg, P, and Zn; serum TBA was negatively (P < 0.05) correlated with hair and serum Cu and fecal Na and positively (P < 0.05) correlated with Correlation between ionomics profiles and fecal significant metabolites. The correlation between ionomics profiles and fecal significant metabolites were described in Fig. 3. Hair Na and K were negatively (P < 0.05) correlated with inosine; hair Cu was negatively (P < 0.05) correlated with putrescine, 2-aminobutyric acid, glucose-6-phosphate, mannose-6-phosphate, inosine, 2-methyl-butanedioic acid, fumaric acid, and oxalic acid; serum Ca, Mg, and P were negatively (P < 0.05) correlated with arginine, homoserine, ornithine, fructose-6-phosphate, 9-(Z)-Octadecenoic acid, 9,12-(Z,Z)-Octadecadienoic acid, 2-hydroxyglutaric acid, and pantothenic acid; fecal Cu was negatively (P < 0.05) correlated with methionine, malic acid, pantothenic acid, and uracil. The significant fecal metabolites (see Supplementary Table S2) which correlated with ionomics profiles were used for further metabolic pathways and metabolite set enrichment analysis (Fig. 4). The hair Cu was negatively correlated with nucleotide sugars metabolism, starch and sucrose metabolism, aspartate metabolism, phenylalanine and tyrosine metabolism, and mitochondrial electron transport chain pathways; serum Ca, Mg, and P were negatively correlated with urea cycle, arginine and proline metabolism, and α-linolenic acid and linoleic acid metabolism pathways; fecal Cu was negatively correlated with β-alanine metabolism, betaine metabolism, malate-aspartate shuttle, and pantothenate and coenzyme A (CoA) biosynthesis pathways.

Discussion
This study explores a new approach of ionomics-metabolome association analysis to investigate the potential relationship between ionomics profiles in hair, serum and feces and differential metabolites in serum and feces. It has been previously found that a high Cu diet promoted the growth rate of pigs 13,17 . In our study, we observed that dietary 300 mg·kg −1 copper enhanced ADG and ADFI during d 14 to 28, but ADG and the G:F value decreased during d 29 to 40 of piglets, previous study showed that dietary 300 mg·kg −1 copper can increase serum GH concentrations in weaning pigs 18 , which suggesting that dietary high levels of copper can promote short-term growth. Several studies have revealed that the positive effects of high-grade copper in the feed of piglets are Figure 2. Correlation between ionomics profiles and serum biochemical parameters. The correlation which exited statistical significant were presented. The red represents a positive correlation (P < 0.05), the blue represents a negative correlation (P < 0.05), and the white shows that the correlation was not significant (P > 0.05). * , ** significantly different among each copper diet, * denote P < 0.05, ** denote P < 0.01. www.nature.com/scientificreports/ Figure 3. Correlation between ionomics profiles and fecal significant metabolites. The correlation which exited statistically significant were presented. The blue represents a negative correlation (P < 0.05), and the white shows that the correlation was not significant (P > 0.05).

Figure 4.
Correlation between ionomics profiles and metabolic pathways. The correlation which exited statistically significant were presented. The blue represents a negative correlation (P < 0.05), and the white shows that the correlation was not significant (P > 0.05). www.nature.com/scientificreports/ mainly growth promotion 19,20 and antibacterial activity 13 , higher nutritional levels of Cu (as CuSO 4 ) at levels of 100 to 250 mg·kg −1 improved growth performance in young pigs 13,17 . Results from our research seemed inconsistent with the results in the above research, and may have been due to the toxic effects of dietary 300 mg·kg −1 copper adversely influencing productive performance of suckling piglets. In our study, dietary 6 mg·kg −1 copper seems unable to meet the nutritional needs of suckling piglets when antibiotics are withdrawn from creep feed because the G:F value decreased compared with the 20 mg·kg −1 copper diet, and the diarrhea rate significantly increased compared with the 300 mg·kg −1 copper diet. The US National Research Council (NRC) has suggested dietary requirements for 5 to 25 kg nursery pigs and growing pigs of approximately 3 to 6 mg·kg −1 copper 13 . The determination of copper content was 5.9 mg·kg −1 in feed of our LC diet. Therefore, the reason for the G:F results in our study may be the increased diarrhea incidence, which in turn affects piglets' weight gain in the 6 mg·kg −1 copper diet. In our study, when piglets were fed a 20 mg·kg −1 copper diet, they tended to gain more and eat less among the three diets across the overall experimental period, which would be adequate to meet the suckling piglets' requirements.
Hair mineral analysis has become an interesting diagnostic tool in the assessment of health and nutritional status 21,22 . In our study, of the 13 elements tested, six elements (Mg, Na, K, P, Cu, and Mn) prominent concentrations changes occur between the three treatment diets. We observed the significant increased concentrations of Na and K, and decreased concentration of Mg and Mn in 300 mg·kg −1 than 20 mg·kg −1 copper diet. Previous studies examined hair trace element contents in women with type 2 diabetes (T2D), Alzheimer's disease (AD) patients, and prostate cancer (PC) patients, interestingly, the significantly elevated Na, K and decreased Mg, Mn were associated with T2D 23 , AD 24 and PC 25 . Our results in hair were consistent with the above studies, which suggested that dietary 300 mg·kg −1 copper may increase the incidence of several diseases of suckling piglets. In our study, the hair Cu concentration significantly increased in 300 than 6 mg·kg −1 copper diet, which shows the same trend variations as dietary copper levels. The high level of hair Cu appeared to be associated with the risk of PC 16,25 , also suggested that dietary 300 mg·kg −1 copper may increase the risk of the development of diseases in piglets. As previously reported, human hair as an excretory system for trace metals, and demonstrated longterm stability of nutritional status 26 , and considering that concentrations of these elements are reported to be correlated with the diagnosis of several diseases 16,27 . These results revealed that the variation trend of Na, K, Mg, Mn and Cu concentrations in hair of suckling piglets can reflect the adverse effects of 300 mg·kg −1 copper diet on the health of piglets to some extent.
Compared with the significant changes in the concentrations of elements in hair, only three elements (Mg, P, and Cu) changed in serum and feces in our study. The results showed that the concentrations of most elements in serum were not directly affected by dietary copper. There have been many reports suggesting that imbalance of several elements in serum appeared to be one of the risk factors of many complex diseases 16 . To further understand the effects of dietary copper on the elemental homeostasis, we analyzed the correlations between each element (see supplementary results, Fig. S1). In the current study, the correlation between Na, K and Cu, Mn, Zn disappears in hair from 20 to 300 mg·kg −1 copper diet (Fig. S1b,c). These results indicated that the concentrations of Na and K, which increased in 300 mg·kg −1 copper diet affected its correlations with Cu, Mn, and Zn. As is well known, trace elements such as Cu, Zn, and Mn are essential for normal growth, disease resistance, production and reproduction in farm animals 28 . This also indicated that with extension of feeding time (d 29 to 40), instead of promoting growth, dietary 300 mg·kg −1 copper it is unfavorable to piglet growth. Combined with these findings, the adversely influence of dietary 300 mg·kg −1 copper on productive performance could be reflected by the correlations between hair Na, K and Cu, Mn, Zn of suckling piglets. Noteworthy changes were also observed in the correlations between macroelements and microelements or toxic elements with increased copper concentrations in the diet. In our study, the positive correlations were increased in 20 than 300 mg·kg −1 copper diet. This result showed the occurrence of altered interdependencies between the elements in serum, which reflects the change in elemental homeostasis. Previously, it was shown that the absorption, utilization, and excretion of many trace elements in animals are greatly affected by other trace elements 28,29 . These results suggest that competitive between high level copper and trace elements can result in trace element deficiencies, thus dietary 20 mg·kg −1 copper can maintain the elemental homeostasis in piglets due to maintenance of the interactions between elements.
To understand the relationship between the ion balance and suckling piglets' health status, correlations between ionomics profiles and serum biochemical parameters were analyzed (Fig. 2). In our study, hair Na and K were positively correlated with T-AOC and negatively correlated with TNF-α. The results showed that there may be a potential link between hair Na and K concentrations and serum T-AOC and TNF-α concentrations. Our previous study indicated that TNF-α was decreased and T-AOC was increased in 300 than 20 mg·kg −1 copper diet 8 (Supplementary Table S1). These results suggested that changes of Na and K concentrations in hair can reflect the effects of 300 mg·kg −1 copper diet on serum inflammatory response and antioxidant capacity of suckling piglets to some extent. The negative correlations between hair Cu with serum MDA and TBA were also observed in our study. The MDA content reflects the severity of body exposure to free radicals, which are crucial biomarkers in the oxidative stress process 30 . In our previous study, the serum MDA was decreased in 20 mg·kg −1 copper diet compared with 6 and 300 mg·kg −1 copper diet, suggesting that dietary 20 mg·kg −1 copper could effectively protect tissues from oxidative damage 8 (Supplementary Table S1). From these results, it seems that there is a certain connection between increased hair Cu concentrations and oxidative stress induced by dietary 300 mg·kg −1 copper. In our study, fecal Cu concentration was positively correlated with serum GH, and the increased serum GH concentration in 300 mg·kg −1 copper diet was also observed (Supplementary Table S1). Consistent with the previous study that high copper concentrations (100 to 300 mg·kg −1 ) can increase the serum GH concentration of weaning pigs 18 . It was believed that the growth-promoting effect of copper was related to the GH axis, and might be generated by the stimulation of GH secretion 31  www.nature.com/scientificreports/ Cu and serum GH. The concentrations of Zn in hair and feces were positively correlated with serum MDA and T-AOC in current study. Zn has the function of stabilizing cell membrane structure and protecting free radicals from oxidative damage 32,33 , this confirmed the above correlations. Results suggested that Zn concentration could reflect the oxidative stress state of suckling piglets. As we know, fecal metabolites reflect the final status of animal digestion, absorption, and metabolism of feed nutrients 8 . Our previous analysis of significant fecal metabolites showed that the capacity of dietary monosaccharide and protein absorption decreased, and the concentration of organic acids was increased in suckling piglets fed with 6 mg·kg −1 Cu diet (see supplementary Fig. S2), these suggest that 6 mg·kg −1 Cu supplementation harms the health of piglets when antibiotic withdrawn from the feed 8 . In this study, we are concerned about changes in the composition of elements in hair, which reflect the body's metabolism changes 34 . The correlation and enrichment analysis showed that the Na and K in hair which significantly affected by dietary copper, were negatively correlated with fecal inosine (Fig. 4), consistent with the result that hair Na and K were negatively correlated with serum TNF-α (Fig. 3), due to the inosine is a purine metabolite and has a systemic anti-inflammatory effect 35 . The hair Cu was significantly increased in the 300 than 6 mg·kg −1 copper diet and negatively correlated with phenylalanine and tyrosine metabolism pathways (Fig. 4), dopamine β-hydroxylase (DBH) and phenylalanine hydrolase are two key enzymes in these pathways which its cofactor is copper 36,37 . The hair Cu was negatively correlated with the mitochondrial electron transport chain pathway (Fig. 4). Various enzymes in this pathway use copper as a cofactor, such as a cytochrome c oxidase and nicotinamide adenine dinucleotide (NADH) dehydrogenase [36][37][38] . The activity of the electron transport chain is related to the generation of reactive oxygen species (ROS) and the body's redox state 39,40 . In this study, dietary 300 mg·kg −1 Cu enhanced the mitochondrial electron transport chain pathway, which promoted the formation of ROS and affecting the redox status of piglets, these verified by the results of negative correlation between hair Cu and serum MDA (Fig. 2).
The fecal Cu content mainly comes from the accumulation of unabsorbed copper in the diet. In our study, fecal Cu was negatively correlated with the betaine metabolism pathway. Betaine is an important methyl donor which provides methyl groups is mainly catalyzed by betaine homocysteine transferase (BHMT), a cytoplasmic enzyme that relies on zinc activation 41 , due to the antagonism between copper and zinc 28 , suggested that high-level dietary copper hinders the absorption of zinc to a certain extent, and inhibits the activity of BHMT as well as the function of betaine methyl donor. In the process of betaine producing methionine, homocysteine is also a substrate for the enzyme action of BHMT. When the activity of BHMT decreases, the concentration of homocysteine in the blood rises, which has a certain relationship with vascular disease, thrombosis, and renal dysfunction 42,43 , these suggested that dietary 300 mg·kg −1 copper inhibited the methyl supply capacity of betaine and further affected protein biosynthesis of suckling piglets. These suggested that 300 mg·kg −1 copper did not support the methyl supply capacity of protein biosynthesis, which further devoid of integral health benefits for suckling piglets. Our previous study found that the concentration of pantothenic acid was decreased in the 300 mg·kg −1 copper diet 8 . In our study, serum Ca, Mg, P concentrations were negatively correlated with urea cycle. The urea cycle is the body's way of converting toxic ammonia into urea. The concentration of urea found in blood can assess how well the kidneys are functioning 44 . These results suggested that serum Ca, Mg, P concentrations may be associated with renal function of suckling piglets, which also verified the changes of serum BUN between 20 and 300 mg·kg −1 copper diet (supplementary Table S1).

Conclusions
Our study suggested that a correlation exists between the changes in certain elements concentrations of hair, serum, and feces and inflammatory response, oxidative stress and antioxidant capacity of suckling piglets when fed with different copper levels in diets. In particular, significantly changed Na, K, Mg, Mn and Cu concentrations in hair reflect the adverse effects of dietary 300 mg·kg −1 copper of suckling piglets, and dietary 20 mg·kg −1 copper maintains the elemental homeostasis in piglets due to maintenance of the interactions between elements. Our results may benefit people to understand the heavy metal elements posing a critical concern to human and animal health from ion interactions and metabolic homeostasis perspective.

Materials and methods
Ethics statement. All  Animal, housing, diets and sampling. The experiments were designed as described in our previous study 8 . A total of 180 piglets (1.11 ± 0.18 kg BW) from 18 multiparous Suhuai sows (second pregnancy) were sorted into blocks by their anticipated farrowing dates. Eighteen litters piglets were randomly assigned to three treatments (6 litters/treatment, 10 piglets per litter), each treatment included three replicates. The treatments (factors) included three levels of copper supplementation from Cu sulfate (CuSO 4 ) according to our previous study 8 : (1) 6 mg·kg −1 copper (LC) diet, containing no supplemental copper; (2) 20 mg·kg −1 copper (CON) diet; or (3) 300 mg·kg −1 copper diet (HC). In each litter, all piglets were selected based on similar body weight (BW), BW and sex were balanced among the piglets, and all piglets were individually weighed within 72 h after farrowing. The piglets were trained to feed when 7 d old with a prefeeding period of 7 to 14 d, and the animal trials were conducted over 26 days (14-40 d), and the corn/soybean based diets were supplied throughout the experiment, meeting the nutritional requirements of the NRC 45 (Table 3). The piglets were housed together with their sow during the experimental period, and with free access to creep feed and water, all piglets were hindered from having access and eating the sow feed 8  Piglet BW and creep feed consumption were used to calculate ADG and ADFI. The diarrhea rate of piglets was recorded daily and calculated as follows: Diarrhea rate (%) = the number of pigs with diarrhea × diarrhea days/ (the total number of pigs × experiment days) × 100%, in which the "number of pigs with diarrhea" was defined as the number of piglets with diarrhea was observed each day 46 . Three litters were chosen within each treatment at 40 d. Feces, blood, and hair samples were collected from four piglets/litter, selected based on average BW (half male and female). Hair samples of the head, back, and buttocks of the piglets were collected and mixed; Blood samples were collected from the anterior vena cava of piglets and stored in glass tubes with no anticoagulant and were allowed to clot at 4 °C before the serum harvest by centrifugation (15 min at 3, 500 g); serum, hair and fecal samples were stored at − 80 °C for subsequent analysis 15 . Ionomics analysis. Thirteen elements, including macroelements (Ca, Mg, Na, K, P), microelements (Fe, Cu, Mn, Zn, Cr), and toxic-elements (Pb, Al, Ni) were measured in hair, serum, and feces using inductively coupled plasma optical emission spectrometry (ICP-OES) (PerkinElmer, USA). Hair (200 μg), serum (1 mL), and dried feces (500 μg) were placed in a tube with 10 mL of a mixture of nitric acid (guaranteed reagent, GR) and perchloric acid (GR) (3:1 v/v). After digestion overnight, tubes were heated from 100 °C to 240 °C over approximately 3 h, and the resulting digests were brought to constant volume with double distilled-deionized water 15,47 . The standard liquid of Ca, Mg, Na, K, and P (1000 µg·mL −1 ) were mixed to prepare 5 mL mixed standards with 0.5 mol·L −1 HNO 3 . Take 0.5 mL mixed standard of Fe, Cu, Mn, Zn and Cr (1000 µg·mL −1 ), constant volume to 5 mL secondary mother liquor (100 µg·mL −1 ), then using secondary mother liquor prepare 10 mL standard with 0.5 mol·L −1 HNO 3 . Take 0.5 mL mixed standard of Pb, Al, and Ni (1000 µg·mL −1 ), constant volume 2 times and prepare 5 mL secondary mother liquor (10 µg·mL −1 ), then prepare 10 mL standard with 0.5 mol·L −1 HNO 3 . According to the instrument software settings, the determination of each standard of solution's absorbance value, each sample determination of repeated three times, get the element of the standard curve, according to the standard curve of the conversion of each element contained in the samples.